Despite advances in our understanding of neuronal responses to cellular stress, interventions for acute neuronal injury remain elusive. One novel avenue of investigation is to identify changes in neuronal metabolism incurred as a consequence of cerebral ischemia. AMP-activated protein kinase (AMPK) is a protein kinase that plays a key regulatory role in energy metabolism in both the brain and peripheral tissues. AMPK is activated via phosphorylation in times of energy demand, as monitored by increasing AMP and declining ATP levels. AMPK reduces fatty acid, cholesterol, and protein biosynthesis and increases catabolic, ATP generating pathways. Recently, numerous stimuli including hypoxia, ischemia/reoxygenation injury, NAD, peroxynitrite (ONOO) and nitric oxide (NO) have been shown to activate AMPK suggesting that AMPK plays a critical role in the response to oxidative stress. We have shown that robust activation of AMPK occurs after middle cerebral artery occlusion (MCAO). Interestingly, this stroke-induced elevation in AMPK is detrimental, as pharmacological inhibition of AMPK is dramatically neuroprotective. In aim 1we will test the hypothesis that reduction in AMPK activation leads to sustained neuroprotection and functional improvement after stroke. The effects of pharmacological agents that decrease AMPK activation will be examined. Aim 2 will evaluate the response to pharmacological activation of AMPK after MCAO. In Aim 3 we will examine animals with targeted deletions of the catalytic subunit of AMPK to confirm our pharmacological data and determine the isoform responsible for the neurotoxicity seen after MCAO. In Aim 4 we will examine the hypothesis that AMPK activation leads to neuronal damage specifically via activation of neuronal NOS (nNOS). Elucidation of the role of AMPK in ischemia may lead to the development of novel treatments for stroke. Relevance: Stroke is the leading cause of disability in the U.S. Interfering with metabolic pathways could prevent or delay cell death by reducing energy demand in the damaged brain. Delaying cell death could prolong the "therapeutic window" in stroke. A multi-faceted approach is clearly needed to develop efficacious neuroprotective agents that will benefit stroke patients. Manipulation of AMPK levels represents a novel approach to neuroprotection.